Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum

Iddar, Abdelghani; Valverde, Federico; Serrano, Aurelio; Soukri, Abdelaziz

doi:10.1016/s1046-5928(02)00032-3

Cited by 52 publications

(44 citation statements)

References 24 publications

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“…2 and 3). GSM1, GSM2, GSM3, and GSM4 showed no growth on acetate, which was expected, since the GapN reaction operates irreversibly in the glycolytic direction but not in the gluconeogenetic direction (51).…”

Section: Design Of a C Glutamicum Strain With Atp-neutral Glycolysismentioning

confidence: 57%

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Reddy

Lindner

Wendisch

2015

Appl Environ Microbiol

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Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADPdependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154 -7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-II D213G and thus by coupling to electron transport phosphorylation (ETP).A TP generation occurs naturally by substrate-level phosphorylation (SLP), electron transport phosphorylation (ETP), photophosphorylation, and decarboxylation phosphorylation. The Embden-Meyerhof-Parnas (EMP) pathway of glycolysis supplies ATP by SLP in the absence of terminal electron acceptors or anaerobic conditions, as well as supplying direct precursors for biomass formation (1). Bacteria and archaea possess one or more of multiple biologically feasible routes for glucose catabolism, such as the EMP pathway, the Entner-Doudoroff (ED) pathway, and the phosphoketolase pathway, which yield zero to 3 ATP molecules per glucose molecule and exist in different variants. In natural habitats, a trade-off between the rate and the yield of ATP production is observed for heterotrophic organisms; faster but less efficient ATP production may be advantageous under conditions characterized by a low abundance of growth substrates (2, 3). For example, Zymomonas mobilis ferments suga...

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Section: Design Of a C Glutamicum Strain With Atp-neutral Glycolysismentioning

confidence: 57%

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Reddy

Lindner

Wendisch

2015

Appl Environ Microbiol

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“…Essentially all other genes whose proteins are assigned to function downstream of acetyl-CoA in the formation of butyrate are moderately downregulated (two-to fivefold) under these conditions. There are two other enzymes that are activated by O 2 and for which a central role in carbon metabolism is apparent (Table 2): one is the nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GapN), catalyzing the NADP-dependent oxidation from glyceraldehyde-3-phosphate to 3-phosphoglycerate (28). Supporting its functional role during glycolysis, genes encoding the enzymes responsible for the subsequent reactions in this pathway (triosephophateisomerase, phosphoglyceromutase, and eno- Table 2).…”

Section: Resultsmentioning

confidence: 99%

The Role of PerR in O₂-Affected Gene Expression ofClostridium acetobutylicum

et al. 2009

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In the strict anaerobe Clostridium acetobutylicum, a PerR-homologous protein has recently been identified as being a key repressor of a reductive machinery for the scavenging of reactive oxygen species and molecular O 2 . In the absence of PerR, the full derepression of its regulon resulted in increased resistance to oxidative stress and nearly full tolerance of an aerobic environment. In the present study, the complementation of a Bacillus subtilis PerR mutant confirmed that the homologous protein from C. acetobutylicum acts as a functional peroxide sensor in vivo. Furthermore, we used a transcriptomic approach to analyze gene expression in the aerotolerant PerR mutant strain and compared it to the O 2 stimulon of wild-type C. acetobutylicum. The genes encoding the components of the alternative detoxification system were PerR regulated. Only few other targets of direct PerR regulation were identified, including two highly expressed genes encoding enzymes that are putatively involved in the central energy metabolism. All of them were highly induced when wild-type cells were exposed to sublethal levels of O 2 . Under these conditions, C. acetobutylicum also activated the repair and biogenesis of DNA and Fe-S clusters as well as the transcription of a gene encoding an unknown CO dehydrogenase-like enzyme. Surprisingly few genes were downregulated when exposed to O 2 , including those involved in butyrate formation. In summary, these results show that the defense of this strict anaerobe against oxidative stress is robust and by far not limited to the removal of O 2 and its reactive derivatives.

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“…These results led us to anticipate difficulties in exploiting the endogenous gapB gene for glucose catabolism, probably due to an intracellular environment in which NAD ϩ is more abundant than NADP ϩ (15). On the other hand, nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN; EC 1.2.1.9), which catalyzes the irreversible oxidation of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP ϩ to NADPH, is found in photosynthetic eukaryotes (10,21) and in some Gram-positive bacterial species, such as Streptococcus (1,4,6,9) and Clostridium species (8). In this work, we attempted to use the heterologous Streptococcus mutans gapN gene to engineer C. glutamicum.…”

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confidence: 99%

Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l -Lysine Production

Takeno

Murata

Kobayashi

et al. 2010

Appl Environ Microbiol

105

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A sufficient supply of NADPH is a critical factor in L-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP ؉ to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an L-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an L-lysine producer was evaluated. The amounts of L-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ϳ70% on glucose, ϳ120% on fructose, and ϳ100% on sucrose, indicating that the increased L-lysine production was attributed to GapN. These results demonstrate effective L-lysine production by C. glutamicum with an additional source of NADPH during glycolysis.

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Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum

Cited by 52 publications

References 24 publications

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

The Role of PerR in O₂-Affected Gene Expression ofClostridium acetobutylicum

Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l -Lysine Production

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Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum

Cited by 52 publications

References 24 publications

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

The Role of PerR in O2-Affected Gene Expression ofClostridium acetobutylicum

Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l -Lysine Production

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The Role of PerR in O₂-Affected Gene Expression ofClostridium acetobutylicum